From the Fourier transform infrared, a stretching vibration of the aluminosilicate tetrahedral was observed, which indicated the success of geopolymerization. The prepared geopolymers showed a high capability of MB adsorption from an aqueous solution. The adsorption process was best suited and explained using the Langmuir isotherm model with a maximum adsorption capacity of 666.7 mg/g for the GEO-MSWBA (at 25°C) and 769.2 mg/g for the GEO-MSWFA (at 35°C). The positive value of the enthalpy (ΔHo) for the GEO-MSWBA suggested the reaction favored endothermic reaction while the negative value of entropy (ΔSo) indicated a solid/liquid random interaction. On the other hand, the negative ΔHo value for the GEO-MSWFA indicated the reaction followed an exothermic reaction causing energy to be released, the positive ΔSo value indicated a good affinity at the solid-liquid surface. The overall negative value for Gibbs free energy (ΔGo) for both adsorbents suggested the adsorption was spontaneous and feasible. It was also inferred that n- π interaction, direct and indirect hydrogen bond, and electrostatic interaction between the MB and the prepared geopolymers facilitated the adsorption process. The current study shows that the GEO-MSWBA and the GEO-MSWFA have a great potential of removing MB as a cationic dye from water without performing any sort of laborious pretreatments.In this study, municipal solid waste bottom ash (MSW-BA) and fly ash (MSW-FA) were used as a source of aluminosilicate to prepare geopolymer (GEO) adsorbents (GEO-MSWBA and GEO-MSWFA) for the removal of methylene blue (MB) from water. The effects of temperature, pH, and initial concentration on the MB adsorption onto GEO-MSWBA and GEO-MSWFA were evaluated. The adsorption isotherms parameters and thermodynamics were also determined. Detailed physical and chemical characterizations of the prepared adsorbents were carried out to further understand their impact on MB adsorption. The results from the scanning electron microscopy revealed a uniform granule-sphere like structure on both prepared geopolymers, which would facilitate the MB adsorption onto the adsorbents. The X-ray diffraction allowed observation of the microstructural transformations that occur after the alkaline activation. The surface areas of the GEO-MSWBA and the GEO-MSWFA were recorded as 32.78 m The treatment of major water pollutants such as toxic metals, drugs, and dyes has become one of the major environmental concerns in the scientific community , 2. VariBasic dyes are one of the common types of dyes that are generally used around the world including methylene blue (MB). Various studies have highlighted that overtime exposure to MB can cause severe health impact on humans, such as tissue necrosis, jaundice, and vomiting . There a4 and SiO4 tetrahedral, which are formed due to the breakdown of Si-O-Si and Si-O-Al bonds. These AlO4 and SiO4 are connected by oxygen corners with a typical 1:3 ratio of Si/Al, which are dissolved in an alkaline medium [2+, Cr3+, Cd2+and Pb2+ [Recent studies have highlighted the potential use of geopolymer (GEO) as adsorbent materials due to their rapid strength, environmental friendliness, ion exchange capacity and effectively solidifying toxic waste . GEO is e medium . GEOs cae medium . Recent e medium –31. GEOse medium . GEOs are medium . Recentland Pb2+ , 32, 33.and Pb2+ , 34–42.This paper reports on the application of geopolymers synthesized from incinerated municipal solid waste ashes (GEO-MSWBA and GEO-MSWFA) on the removal of MB from water. The effects of temperature, pH, and initial MB concentration on the MB adsorption onto the GEO-MSWBA and GEO-MSWFA were evaluated. The adsorption isotherms parameters and thermodynamics were also determined. Detailed physical and chemical characterizations of the prepared geopolymers were carried out to further understand their impact on MB adsorption using Fourier transform infrared (FTIR), scanning electron microscopes-energy-dispersive X-ray spectroscopy (SEM-EDX) and X-ray diffractometry (XRD). Four different adsorption isotherm models, namely Langmuir, Freundlich, Temkin, and Dubinin-Radushkevich were studied in order to determine the mechanisms and efficiency of the adsorption process.Hydrochloric acid  and hydroxide (NaOH) extra pure pellets were obtained from Scharlu Barcelona (Spain). Sodium metasilicate was obtained from Sigma-Aldrich from (Germany). Extraction solutions were prepared with deionized water obtained from a Milli-Q system, 0.20 μm resistivity .oC for 24 h, ground at different particle sizes, and sieved through a standard sieve. Then the samples were kept in clean and isolated glass bottles. A particle size of less than 250 μm was used for the geopolymer preparation. Sodium silicate solution (Na2SiO3) was prepared by dissolving Na2SiO3 in water and heated in an electric stove at 175°C.To obtain a homogenous and representative sample, replicate samples were collected at different periods. MSW-BA and MSW-FA were freshly collected in a 5 kg representative sample from a local incinerator (Qatar Company: Domestic Solid Waste Management Centre (DSWMC)–Doha, State of Qatar). The permission of the sample collection was already granted by the Company. We confirm that the field studies did not involve endangered or protected species. To minimize the change in the physiochemical characteristics due to natural weathering, the ashes were immediately dried at 100 2SiO3 solution was then added to the slurry specimen and stirred until a homogenous solution was obtained. The resulting paste was placed in an oven at 65°C for 72 h until the mixture was completely dry. Excess NaOH was removed from the geopolymer mixture paste by continuously washing with deionized water. The specimens were then crushed and sieved to obtain uniform smaller particles for the experiment. A particle size of less than 250 μm was used to perform the geopolymer characterizations and the adsorption studies. The mixed proportion of the geopolymer specimens were 60% ash (MSW-BA or MSW-FA), 16% NaOH, and 24% Na2SiO3 by weight [Geopolymer adsorbents were prepared by adding 30 g of the MSW-BA or MSW-FA to 8 mL of 5 M NaOH. The mixture was mixed for 10 minutes at room temperature to allow ions leaching and formation of a slurry mixture. Nay weight . The pre−1. The crystalline phase of the prepared geopolymers was analyzed via XRD . The morphology and microstructure of samples were visualized and analyzed by SEM . The pore size distribution and surface area were obtained using the Brunauer-Emmett-Teller (BET) analyzer .The main functional groups of the prepared geopolymers were characterized by FTIR . FTIR analysis was carried out in the range of 4000–400 cmTwo way ANOVA was performed using Excel 2018 for the effect of pH, temperature, and the initial concentration on the MB adsorption process onto GEO-MSWBA and GEO-MSWFA.All experiments were performed using the batch process. Four linearized forms of the isotherm models were used, namely Langmuir, Freundlich, Dubinin-Radushkevish, and Temkin to evaluate the adsorption capacity. A fixed amount of the geopolymer GEO-MSWBA or GEO-MSWFA (0.05 g) was added to a 50 mL of the MB solution at different pH values, temperatures, and initial MB concentrations, and placed in a temperature-controlled shaker at 130 rpm . To ensure quality, control blank and two trials were prepared. Each solution concentration was measured under 663 nm  using a 0 and Ceq are the initial and equilibrium of MB concentrations (mg/L), respectively.Where, CThe adsorption capacity was calculated using e is the amount of MB adsorbed by the geopolymer (mg/g), V is the volume (L), and m is the mass of the prepared geopolymer (g).Where qoC for 48 h at various pH values . The pH values were adjusted using a pH meter (Jenway 370). 0.1 M sodium hydroxide (NaOH) and 0.1 M hydrochloric acid (HCl) were used to adjust the pH value. Different initial MB concentrations were studied . The MB adsorption was also studied at various temperatures . The removal efficiency and adsorption capacity were measured after the system reached equilibrium, which was 48 h.A 0.05 g of the prepared geopolymer was shaken with a 50 mL of the 1000 mg MB /L solution at 25 It was noticed that the surface of the MSW-BA and the MSW-FA after geopolymerization became rougher, which indicated the decomposition of clusters. The irregular shape in both GEO-MSWBA and GEO-MSWFA can be related to the amorphous composition of material. The hydrous metal oxide formation changed the material surface further by making it coarser, non-homogenous, with cavities and protuberance. The more porous structure indeed facilitated the adsorption phenomenon for liquid and solid phases. The random arrangement of the structure further helped the adsorption of MB by preventing the escape of ions between the particles by creating a more network-like structure. Similar results were observed in , 49. Chiθ), various chemical compositions were observed. The XRD allowed observation of the microstructural transformations that occur after the alkaline activation. In both Figs 3) and quartz (SiO2). While the peak at 32.5° indicated the presence of calcium silicate hydrate phase in both geopolymers [6Si12O13) and quartz. The intensity of the reflection peaks of calcite is higher in the GEO-MSWBA. This could be related to the presence of higher calcium hydroxide content surrounding calcium silicate hydrate due to the higher CaO content.On the other hand, the diffractrograms of the GEO-MSWBA and the GEO-MSWFA were also studied as shown in Figs polymers . Furtherpolymers  studied polymers  fly ash 2/g and 4.5 m2/g, respectively. The high surface area corresponds to perhaps an improvement in surface area of the GEO-MSWBA to adsorb MB more effectively. Ge et al. [2/g. While Tang et al. [2/g.The surface area can affect the adsorption rate as well as the uptake capacity. The BET surface areas of the GEO-MSWBA and the GEO-MSWFA were 32.78 me et al.  preparedg et al.  stated m3/g, and 0.03 cm3/g for the GEO-MSWFA. The pore size of the GEO-MSWBA was 64.8 nm while for the GEO-MSWFA was 26.7 nm. When the values were compared with other prepared geopolymers, for instance, Liu et al., 2016, the pore volume and the pore size of the prepared geopolymer were 0.070 cm3/g and 19.62 nm, respectively. Similarly, Lee et al. [3/g and pore size to be 8.98 nm. Kara et al. [2/g. Furthermore, Rasaki et al. [2/g, and pore size was 11.5 nm.While the total pore volume was also observed to be higher for the GEO-MSWBA which was 0.14 cme et al.  found tha et al.  prepared-1, which indicated the presence of Al-Fe-OH while the bending at 975 cm-1 indicated the vibration of Al-Al-OH. The vibration at 1115 cm-1 indicates the presence of Si-O. Another stretch can also be observed at 1138 cm-1, which shows C-O presence. The peak observed between 2900–2800 cm-1 shows the C-H aldehydic functional group and the peak at 3381 cm-1 indicates the presence of the O-H bond. In contrast, the spectrum of the GEO-MSWBA indicates the presence of stretching vibration of the aldehyde functional group between 2800 cm-1 and 2900 cm-1, furthermore, the absorption band corresponds to the stretching vibration of aliphatic groups C-H. The presence of a peak at ~1460 cm-1 signifies the presence of sodium carbonate [-1 is located for O-H bending [-1 attributes to the presence of Si-O bond while the peak at 1000 cm-1 further assures the presence of silica. The peak at 1500 cm-1 indicates the C-O bond. 2940 and 2842 cm-1 indicate the presence of -CH3 asymmetric and -CH2 symmetric stretching vibration respectively. In contrast, the GEO-MSWFA spectrum shows a stretching and deformation vibration at 1640 cm-1 of OH [-1 the stretching vibration owes it to the asymmetric bond of Al-O and Si-O [-1 [-1 indicates the presence of Si-O-Si, which indicates the success of the geopolymerization of both ashes. Furthermore, the absorption band at 2800 and 2900 cm-1 correspond to the stretching vibration of aliphatic C-H. While the peak at around 1400 cm-1 is related to the presence of carbonate. 1410 cm-1 in the GEO-MSWFA was due to O-C-O stretching vibration which indicates the presence of sodium carbonate [-1 associated with asymmetric Al-O-Al/Si-O-Si stretching. While Barbosa et al. [-1 represents the sodium carbonate resulting from the carbonation. It is important to highlight that in both spectra the bands at 984 cm-1 and 677 cm-1 corresponded to the aluminosilicate tetrahedral asymmetric and symmetric stretch vibration, respectively [arbonate . The sig bending . While Fand Si-O . Si-O-SiSi-O [-1 . The strarbonate . A simila et al.  mentioneectively . The presolution of the prepared geopolymers after 24 h of stirring was 10.01, indicating a strong basicity of the prepared geopolymers. The geopolymer reaction caused a negative solid surface charge [One of the important parameters in the sorption process is the effect of pH. Changes in the pH solution impacts on the MB adsorption onto the adsorbent. e charge . The pree) was 55.04 mg/g at 100 mg/L while increased to 190.51 mg/g and 800.87 mg/g at 200 mg/L and 900 mg/L, respectively. On the other hand, a similar trend was followed for the GEO-MSWFA, at 100 mg/L was 75.03 mg/g, which further increased to 199.51 mg/g at 200 mg/L. While by 800 mg/L the amount increased to 781.90 mg/g. It was concluded as the concentration of MB increased the amount adsorbed on the GEOs increased. This was due to the sufficient available pores on the adsorbents. It can be said that MB acted as a driving force to prevail in the resistance to the mass transfer of MB between the MB aqueous solution and the adsorbent. For both prepared geopolymers, the adsorption capacity did not increase sufficiently after 800 mg/L, this is probably because the geopolymer reached the maximum adsorption capacity. A study by Maleki et al. [i et al.  also conTo validate the data of the effect of pH, temperature, and the initial concentration on the adsorption process, two way ANOVA was performed in this study. The probability value  can be defined as a significant level, which leads to the null hypothesis rejection. The ratio between the mean square effect, errors, and the p-value is determined using Fischer Variance (F) . AccordiThe Langmuir adsorption isotherm is one of the common isotherm that is used to describe the equilibrium between an adsorbent and adsorbate. In this model, the adsorbent surface is assumed homogenous and there is no interaction between the molecules adsorbed and the adsorbed surface . On the 2) was close to 1; (0.94–0.99) for the GEO-MSWBA, and (0.92–0.98) for the GEO-MSWFA. This indicated that the MB adsorption onto the GEO-MSWBA and the GEO-MSWFA could be best explained by the Langmuir isotherm. The RL, which represents the separation factor was found to be between 0 < RL < 1 for both adsorbents, showed that the process was energetically favorable. The KL constant, which refers to the affinity between the adsorbate and adsorbent, suggests that there was comparatively stronger binding at 45°C for the GEO-MSWBA, while for the GEO-MSWFA at 25°C showed a high affinity. Furthermore, the Q0 which denotes the maximum adsorption capacity showed that as the temperature increased the maximum capacity decreased for the GEO-MSWBA; 666.7 mg/g at 25°C while 476.2 mg/g at 45°C. However, for the GEO-MSWFA, the maximum capacity increased at 35°C, from 434.7 mg/g at 25°C to 769.3 mg/g at 35°C.Adsorption isotherm illustrates the interaction between adsorbate and the adsorbent occurring at either maximum adsorption capacity or equilibrium . Isotherf value showed an overall increase as the temperature increased 3.42 (mg/g)(L/g)n and 10.31 (mg/g)(L/g)n at 35 and 45°C, respectively, which indicated that as the temperature increased the adsorption capacity also increased. While for the GEO-MSWFA, as temperature increased the Kf value decreased, which indicated that the high temperature did not favor the adsorption process. The n value explains if the adsorption was chemical adsorption or physical adsorption. Less than 1 indicates a chemical adsorption while more than 1 shows the adsorption was a physical process. For the GEO-MSWBA and the GEO-MSWFA, the reaction seemed to follow a physical adsorption. While the value for 1/n displays the reaction was favorable. However, the R2 values for the GEO-MSWBA were low, ranging between 0.74–0.52 while the GEO-MSWFA was between 0.88–98. This suggests that the Freundlich model might not be suitable to describe the adsorption process of the GEO-MSWBA.The Freundlich isotherm assumes that the uptake of MB ion took place on a heterogeneous surface, and the distribution of heat was non-uniform over the surface. From T) showed an increase and decrease as the temperature increases. For instance, the AT value was 0.02 L/g at 25°C and changed to 23.31 L/g at 35°C and 0.11 L/g at 45°C for GEO-MSWBA, while for the GEO-MSWFA, the AT value was 0.05 L/g at 25°C and changed to 33.89 L/g at 35°C and then dropped to 21.57 L/g at 45°C. The heat adsorption constant (BT) showed that the heat adsorption was high at 25°C (114.4 J/mol) and 35°C (96.92 J/mol) for the GEO-MSWBA while for the GEO-MSWFA, it was high at 35°C (174.2 J/mol) and 45°C (146.7 J/mol). Lastly, for the Dubinin-Radushkevich, R2 ranged between 0.27 to 0.76 for 35 and 45°C, which suggested that the model was not suitable to describe the adsorption of MB onto the GEO-MSWBA and the GEO-MSWFA.The Temkin isotherm equilibrium binding constant  of adsorption per mole of the adsorbate (kJ/mol). The ED can be obtained from the The KD value is less than 8 kJ/mol. However, the ion exchange mechanism will be governed when the ED value is between 8 and 16 kJ/mol. For the value of ED greater than16 kJ/mol, the particle diffusion phenomenon will be dominant.The physical adsorption mechanism will be dominant when the ED (per molecule of adsorbate) from the Dubinin-Radushkevich adsorption model was used in finding the most-stable MB adsorbed onto the surfaces of the GEO-MSWBA and GEO-MSWFA. For example, in the MB adsorbed onto the GEO-MSWFA system, the ED was calculated according to the To distinguish between the physical and chemical adsorption of MB ions, the EMB adsorbed on GEO-MSWFA, EGEO-MSWFA and EMB are total energies of the MB ions adsorbed GEO-MSWFA system, the GEO-MSWFA surface, and single MB ions, respectively.Where ED) of the adsorption per mole of the adsorbate (9.71 kJ mol-1 at 25°C); suggesting that the adsorption process is dominated by an ion-exchange mechanism. This variation on ED values for the GEO-MSWFA could be related to the MB adsorption profile onto the surface of the GEO-MSWFA and the MB oxidation and reduction. This also could be explained as once the MB adsorbed on the surface of the GEO-MSWFA may undergo an equilibrium of reduction and oxidation, and the MB configuration could be changed according to the state of MB and the amount of energy required for adsorption ) onto the surface of the GEO-MSWFA as shown in T values are positive for the GEO-MSWBA and the GEO-MSWFA, which means that the adsorption reaction is exothermic (substitution of one or more water molecules by an organic molecule).The Langmuir QT is the adsorption-adsorbent Temkin constant (L/mol), ΔBT is the variation of the adsorption energy (kJ/mol) of Temkin.AHo), Gibbs free energy (ΔGo), and entropy (ΔSo) were determined by using the effect of temperature and van't Hoff equation. The relationship between the adsorption coefficient KL from the Langmuir adsorption isotherm and the temperature is expressed as Eqs Thermodynamic parameters, i.e., enthalpy , ΔSo is entropy change (J/mol.K), and ΔGo is Gibbs free energy change (kJ/mol). KL is the Langmuir adsorption equilibrium constant (L/mol), R is the gas constant (8.314 J/mol.K) and T is the absolute temperature (K).Where Δo becomes more negative with an increase in temperature. The decrease in ΔGo with an increase in temperature also showed that the adsorption process was endothermic. The positive value of enthalpy (ΔHo) for the GEO-MSWBA indicated that the process followed an endothermic reaction, in addition, the negative value of ΔSo indicated solid/liquid random interaction throughout the adsorption process [o) for GEO-MSWFA suggested that the MB adsorption onto GEO-MSWFA had a good affinity at the solid-liquid surface.From the data in  process . While f3+, Mg2+, and K+ and the conjugated structure of the MB molecule might have also caused n- π interaction , which accelerated the mass transfer of MB onto the adsorbate . It can eraction . Furthero for GEO-MSWBA indicated random state at solid-liquid interference throughout the adsorption process of MB while the positive value of ΔSo for GEO-MSWFA suggested that the MB adsorption had a good affinity at the solid-liquid surface. The negative value for ΔGo for both adsorbents indicated that the reaction was spontaneous and feasible. The studies also concluded that the reaction followed an exothermic reaction for the GEO-MSWBA and favored low temperature (25 oC). While the GEO-MSWFA followed an endothermic reaction and was more feasible at a slightly higher temperature (35 oC).This work demonstrated a successful preparation of low-cost adsorbents prepared by geopolymerization. In the present study, the GEO-MSWBA and GEO-MSWFA were prepared using MSW-BA and MSW-FA. The presence of alumino-silicate gel demonstrated the success of geopolymerization. Furthermore, the morphology of the GEO-MSWBA and GEO-MSWFA also transformed from smooth to a rough surface consisting of much more dense pores in addition the SEM graphs also showed an increase in specific surface area as well as morphological amendments. The presence of the Al-O-Al/Si-O-Si functional groups that were also found in FTIR further confirmed a successful geopolymerization. The adsorption process in both adsorbents was best described using the Langmuir model. The maximum MB adsorption capacity was 666.7 mg/g (at 25°C) for the GEO-MSWBA while 769.2 mg/g (at 35°C) for the GEO-MSWFA. The thermodynamic functions such as ΔG°, ΔH°, and ΔS° in the adsorption process were also examined. The negative value of ΔSThe outcome of this study also sheds light on the ability of utilizing local by-products as a highly efficient, potentiated, and inexpensive adsorbents in contrast to other expensive adsorbents offered in the market. It possesses advantages such as high adsorption efficiency, easy separation from the liquid solution, waste minimization, cutback in landfilling practices, and preventing the utilization of virgin raw material. For future studies, the adsorbents shall be further exploited in order to enhance the geopolymer capabilities of effectively removing other cationic dyes from dyeing wastewater or other emerging pollutants and also explore the possibility of utilizing it on a larger scale.


First, a cross-sectional study in the discovery cohort consisting of 1378 participants  from the general population was carried out. To validate and extend the results, two replication cohorts consisting of elderly individuals were studied. In the discovery cohort, we observed a strong gene-age interaction (p = 0.001), determining fasting glucose in such a way that the increasing effect of the risk G-allele was much greater in young (p = 5.9 × 10−10) than in elderly participants (p = 0.805). Consistently, the association of the MTNR1B-rs10830963 polymorphism with fasting glucose concentrations in the two replication cohorts (mean age over 65 years) did not reach statistical significance (p > 0.05 for both). However, in the elderly cohorts, significant associations between the polymorphism and type-2 diabetes at baseline were found. Moreover, in one of the cohorts, we obtained a statistically significant interaction between the MTNR1B polymorphism and the number of pregnancies, retrospectively assessed, on the type-2 diabetes risk. In conclusion, the association of the MTNR1B-rs10830963 polymorphism with fasting glucose is age-dependent, having a greater effect in younger people. However, in elderly subjects, associations of the polymorphism with type-2 diabetes were observed and our exploratory analysis suggested a modulatory effect of the number of past pregnancies on the future type-2 diabetes genetic risk.Gene-age interactions have not been systematically investigated on metabolic phenotypes and this modulation will be key for a better understanding of the temporal regulation in nutrigenomics. Taking into account that aging is typically associated with both impairment of the circadian system and a decrease in melatonin secretion, we focused on the melatonin receptor 1B (MTNR1B)-rs10830963 C>G variant that has been associated with fasting glucose concentrations, gestational diabetes, and type-2 diabetes. Therefore, our main aim was to investigate whether the association between the MTNR1B-rs10830963 polymorphism and fasting glucose is age dependent. Our secondary aims were to analyze the polymorphism association with type-2 diabetes and explore the gene-pregnancies interactions on the later type-2 diabetes risk. Three Mediterranean cohorts ( Currently, there is renewed interest in research into precision nutrition ,2, an exHence, an aspect that has to be analyzed in more detail before tackling a gene-diet interaction study is whether the main effects of a genetic polymorphism are homogeneous per age or if there is heterogeneity. Despite knowing that this is fundamental for its application in precision nutrition ,2,3,8, fThis paper focuses on the study of gene-age interactions of a very important gene related to chronobiology ,16,17,18Circadian rhythms and sleep quality are closely regulated by melatonin, a crucial hormone determining the sleep-wake cycle in humans . HoweverAfter examining the chronological age of participants included in the corresponding published studies, we observed that the associations between the MTNR1B-rs10830963 polymorphism and fasting glucose concentrations were higher in investigations carried out in young populations ,23,25,26n = 2823) of a Spanish Mediterranean population from three defined cohorts were analyzed [2) and the controls were non-obese individuals (BMI < 30 kg/m2) recruited from the same place and without pairing for age and sex. Cases and controls were apparently healthy individuals recruited through advertisements in shopping malls, housewives’ associations, cultural associations and other types of groups from the general population, public and private institutions, educational centers, home contacts, and primary health care centers. The exclusion criteria were being pregnant or breast-feeding, suffering from some infectious/contagious disease, invalidating physical or psychological diseases, cancer, thyroid diseases, Cushing disease, high alcohol intake, or the consumption of other drugs, as previously detailed. In this study, 1378 participants (543 men and 835 women), who had consecutively complete data available for the MTNR1B-rs10830963 polymorphism and fasting glucose concentrations, were analyzed. Of them, 53 (3.8%) were type-2 diabetics. Participants provided informed consent and study protocol and procedures were approved according to the ethical standards of the Helsinki Declaration and by the Human Research Ethics Committee of the University of Valencia, Valencia .In this study, individuals . Finally, another cohort (replication cohort 2) was obtained to analyze the replication of the MTNR1B-rs10830963 SNP associations with fasting glucose and with type-2 diabetes risk in an elderly population, as well as to extend results by undertaking an additional exploratory analysis related to the influence of the number of pregnancies on the type-2 diabetes risk conferred by the polymorphism. We considered this analysis as exploratory, bearing in mind the small sample size in this cohort. This cohort consisted of participants in the PREDIMED Plus-Valencia study. The characteristics of this study have previously been published [Later, in order to check the possible replication of the associations of the MTNR1B-rs10830963 polymorphism with fasting glucose in a more elderly population, as well as to extend the results by testing the association between the MTNR1B-rs10830963 polymorphism and type-2 diabetes, given that prevalence was low in the general population, a replication cohort 1 was selected. This replication cohort consisted of 1001 elderly individuals  participating in the “PREvención con DIeta MEDiterránea” (PREDIMED)-Valencia study. Details of this study have previously been published . BrieflyThe assessment of the participants in the three cohorts was performed by the same researchers using similar protocols. Sociodemographic and clinical variables were obtained through the standardized questionnaires previously used and specifically designed for these cohorts ,62,63. Ap = 0.091, p = 0.900, and p = 0.889, respectively).Genomic DNA in the three cohorts was isolated from white blood cells. DNA quality control was undertaken in the laboratory of the University of Valencia, as previously reported ,62,63. Ip <0.05.Chi-square tests were used to compare proportions and Student’s t and ANOVA tests were applied to compare continuous variables. Crude and multivariable-adjusted models were fitted for each cohort. The MTNR1B-rs10830963 polymorphism was considered codominant (three genotypes) as well as additive , depending on the genetic model analyzed. For the cross-sectional analysis in the discovery cohort, general linear models were used. Firstly, an unadjusted model was used to test the association between the MTNR1B-rs10830963 polymorphism and fasting glucose concentrations. The crude model was then sequentially adjusted for other potential confounders as follows: adjusted for age, sex, and type-2 diabetes and adjusted for obesity. Additional adjustment for other variables  for specific analyses were carried out. When indicated, analyses were stratified for non-diabetic and type-2 diabetic subjects. The estimates were made for both the whole population (men and women jointly) and were stratified by sex to apply the sex/gender perspective to our analyses . To testn = 684 subjects) and the older group . This cohort was relatively healthy, with a very low prevalence of type-2 diabetes (3.8%). The minor allele frequency (MAF) of the variant risk allele “G” was 0.31, similar to other European populations [p = 0.409), nor were any differences detected in the frequency between age groups (p = 0.554) in those less than or greater than 41 years.First of all, the association of the MTNR1B-rs10830963 polymorphism with fasting glucose was analyzed in 1378 subjects from the general population (OBENUTIC study). This cohort was considered the discovery cohort. ulations ,33. No dn = 1378 in the discovery cohort), a statistically significant association was found between the MTNR1B-rs10830963 polymorphism (codominant model) and fasting glucose (p = 0.009) in the unadjusted model. This polymorphism main effect remained statistically significant in the model adjusted for sex, age (as continuous), type-2 diabetes, and obesity (p = 0.015). p-value for the polymorphism in the additive genetic model, adjusted for the above indicated covariates, was calculated (p = 0.005). The homozygous GG were those that had higher fasting glucose in comparison with the CC homozygous and in an intermediate position were heterozygous CG.When considering the whole population  in the discovery cohort were carried out. When only considering non-diabetic subjects in this cohort (OBENUTIC study), the association of the MTNR1B-rs10830963 polymorphism in the codominant (p = 4 × 10−6) as well as in the additive genetic model (p = 6.84 × 10−7) with fasting glucose concentrations in the model adjusted for sex, age, and obesity remained highly significant (p = 6.84 × 10−7). In the model for the whole population (including non-diabetic and type-2 diabetic subjects), the regression coefficient for age was B = 0.43 ± 0.03 mg/dL; p < 0.001, showing an increase of fasting glucose with increasing age. In non-diabetic subjects, the regression coefficient for age was B = 0.31 ± 0.02 mg/dL; p < 0.001. Further adjustment of this model by tobacco smoking (23.5% smokers) did not change the statistical association (p = 4.15 × 10−7). Moreover, after additional adjustment of this model for blood pressure, total cholesterol, and triglycerides, the association between the polymorphism and fasting glucose remained statistically significant (p = 7.0 × 10−6). Finally, on studying the association stratified per sex, in both non-diabetic men and women, the G allele was significantly associated with higher fasting glucose in men (p = 4.7 × 10−5) and in women (p = 0.002).To minimize the possibility that the fasting glucose association might reflect the inclusion within the cross-sectional study samples of subjects with known type-2 diabetes, stratified analyses of the associations in non-diabetic subjects  was studied, including type-2 diabetic subjects and non-diabetics. In a model adjusted for sex, age (as continuous), type-2 diabetes, and obesity, a statistically significant interaction was obtained between age and the MTNR1B-rs10830963 polymorphism (p = 0.001). Even after additional adjustment for diabetes medication, this gene-age interaction remained statistically significant (p = 0.002).First, the statistical significance of that interaction for the whole population (n = 1325) of the discovery cohort. Likewise, a statistically significant gene-age interaction was obtained both in the crude model (p = 0.008) and in those adjusted for age and sex (p = 0.012) and subsequently, for obesity (p = 0.009). In the same way, as mentioned above, to obtain a better estimation of the effects on fasting glucose in non-diabetics, the gene-age interaction analysis was computed only in non-diabetic subjects (n = 1378) and in non-diabetic participants (n = 1325). The polymorphism was analyzed both as codominant (three genotypes) and as additive . The corresponding means by genotype or the regression coefficients (depending on the model), the SE, and the statistical significance for each age-group are indicated. In addition, the p-values for the corresponding interaction terms for each statistical model are shown. All the p-values for the interaction terms between the MTNR1B-rs10830963 polymorphism and the two age groups are statistically significant, indicating a robust consistency for this interaction. Each statistical model in p < 1 × 10−7 for all). In contrast, in the older group, genetic effect size of the MTNR1B-rs10830963 on fasting glucose was slight and did not reach statistical significance . For example, for the whole population (n = 1378) of the discovery cohort, in the fully adjusted model (p3) and considering the polymorphism as additive, we found a statistically significant interaction term between the polymorphism and age (p = 0.001). In subjects aged ≤ 41 years, the regression coefficient for the multivariate-adjusted model was B = 3.00 ± 0.47 mg/dL, indicating a strong effect per G-allele increasing in fasting glucose in this age group (p = 4.61 × 10−10). However, the same model in subjects aged over 41 years revealed a non-statistically significant effect  of the MTNR1B-rs10830963 polymorphisms on fasting glucose concentrations in this group.To better characterize this interaction per age, the dichotomous age variable based on the population mean (41 years) was analyzed. n = 1378). We observed that in the stratified analysis by sex, the p-values for the interaction term between the MTNR1B-rs10830963 polymorphism (as additive) and age (dichotomous in 41 years) determining fasting glucose concentrations in the fully adjusted model including age, type-2 diabetes, and obesity were: p = 0.021 for men and p = 0.032 for women. Thus, the age-interaction effects for men and women were present in both.Furthermore, homogeneity per sex was analyzed in the discovery cohort . For the whole replication cohort 1 (n = 1001) in a model adjusted for sex, age, type-2 diabetes, and obesity, the MTNR1B-rs10830963 G-allele (additive model) presented a small effect size and was not significantly associated with fasting glucose . No significant changes were detected after additional adjustment for smoking or diabetes medication (not shown).Given that this is the first time that interaction per age in the effects of the MTNR1B-rs10830963 polymorphism on determining fasting glucose has been characterized, we wanted to check whether in another elderly population, the non-association of the polymorphism with fasting glucose could be replicated. A replication and extension cohort consisting of 1001 participants from the PREDIMED-Valencia study were analyzed. p = 0.050 in non-diabetic subjects. No significant association with age was found in diabetic subjects (p = 0.401).Given that the prevalence of type-2 diabetes in this replication cohort 1 is high (46.4%) compared with the discovery cohort, we analyzed the association between the MTNR1B-rs10830963 and type-2 diabetes prevalence .p = 0.048. Interestingly, this result shows the effect of the MTNR1B-rs10830963 polymorphism on prevalent type-2 diabetes, even in an elderly population. On analyzing homogeneity by sex, the results may suggest a relatively higher effect in women  in comparison to men  in the model adjusted for age and obesity. However, the sample size is small and no statistical differences between sexes were reached.GG genotype was more prevalent in type-2 diabetic subjects than in non-diabetics. Likewise, in an additive model, the G-allele was significantly associated with type-2 diabetes risk, even after adjustment for sex, age, and obesity: OR: 1.22; 95%CI: 1.01–1.48; p = 0.219). Likewise, no associations were detected when we analyzed non-diabetic participants from this cohort with the same model .p = 0.434) in an additive model adjusted for sex, age, and obesity. We explored whether the number of full-term pregnancies retrospectively reported by women interacted with the MTNR1B polymorphism and the later risk of type-2 diabetes. The number of pregnancies ranged from 0 to 7, with a distribution as follows: none (5.6%), one (10.8%), two (46.0%), three (23.6%), and four or more (14%). In a model adjusted for age and obesity, we detected a statistically significant interaction term between the MTNR1B-rs10830963 polymorphism (additive model) and the number of full-term pregnancies (as continuous), being the estimation for this interaction term: OR: 1.48, 95%CI: 1.01–2.16; p = 0.042, indicating that a higher number of pregnancies in carriers of the G-allele significantly increased the later risk of type-2 diabetes. Considering that these women’s mean age is 67 years, at least 25–35 years had passed by since the end of their fertile age.Regarding associations with type-2 diabetes risk, in replication cohort 2, the sample size was smaller than in replication cohort 1. Therefore, the effects have to be greater to be detected as statistically significant. The MTNR1B-rs10830963 polymorphism did not reach statistical significance for the whole population  from three Mediterranean cohorts, we have been able to demonstrate that the effects of the MTNR1B-rs10830963 C>G polymorphism  on fastiDespite the relevance of age, its impact on the genetic effects has been largely unexplored. For the vast majority of genes involved in glucose metabolism or chronobiology, we do not know whether the magnitude of the genetic effects is constant or whether it varies with age. In an initial Genome-wide association study (GWAS) meta-analysis on more than 35,000 individuals of European descent, a highly significant association between the rs10830963 C>G polymorphism and fasting plasma glucose concentrations was reported . Overallp < 0.0001), so they concluded that “the effect sizes in children and adolescents seem to be stronger than in adults.” This conclusion supports our results regarding the interaction according to age and our observation that the effects are greater in a younger population than in an elderly population.We tested the age-MTNR1B-rs10830963 polymorphism interaction in our discovery cohort and found a strong interaction effect both on considering age as a continuous variable (we detected a step-wise decrease in the genetic effect size at increasing age) and on considering age as a categorical variable (two groups based on the population mean). Therefore, we were able to confirm that the effect size of the MTNR1B-rs10830963 polymorphism on fasting glucose is greater in young people and diminishes in the older population to the point of not detecting statistically significant effects in the older groups. As far as we know, no previous study has specifically reported a statistically significant interaction between the MTNR1B-rs10830963 polymorphism and age in determining fasting glucose concentrations . However, in some studies and meta-analyses, the existence of statistically significant heterogeneity in genetic effect sizes of the MTNR1B-rs10830963 polymorphism on fasting glucose concentrations has been reported ,41. ThatOverall, this gene-age interaction has been masked in large cohorts, including individuals with a wide age range, despite the mean age of those cohorts being elderly, due to the fact that the mean main effect of the polymorphism is relatively high and statistically significant associations of the polymorphism with fasting glucose have been found . This doIn our studies and following the recommendations of applying the gender perspective when generating scientific results , we examOn analyzing the results stratified by sex, we observed that the type-2 diabetes risk results were slightly more significant in women (borderline) than in men. That may be explained in part by considering that this polymorphism has been consistently associated with a higher risk of gestational diabetes in women ,36,38,40It is well established that women with gestational diabetes are at increased risk for future type-2 diabetes . MoreoveIn conclusion, we have a found a statistically significant gene-age interaction between the MTNR1B-rs10830963 polymorphism and chronological age in determining fasting glucose concentrations. This heterogeneity in the association of the MTNR1B-rs10830963 with fasting glucose concentrations illustrates the importance of better understanding of the presence or absence of interactions with age on genetic markers before undertaking specific studies of gene-diet interactions. Likewise, our results suggest that on analyzing the association between the MTNR1B-rs10830963 polymorphism and type-2 diabetes risk, sex-specific variables related to pregnancy in women should be considered.


Sickle cell disease (SCD) is a common but neglected condition in sub-Saharan Africa. Although up to 230,000 babies are born with SCD in the region every year , most coz-score), wasting (a low weight-for-height z-score), and underweight (a low weight-for-height z-score) were 55.4% (54.5–56.4%), 9.1% (8.6–9.7%), and 38.9% (38.0–39.8%), respectively [For the first time in Africa, real-time SCD testing of children between six and 59 months of age (through use of the SickleSCAN™ rapid test) was included in the latest Nigerian Demographic Health Survey . This gaAlthough undernutrition has long been recognised as an important complication of SCD , this stWhile the aetiology of undernutrition in children with SCD is not completely understood (reviewed in reference ), known This study provides important evidence about the frequency of undernutrition in children with SCD in Nigeria. However, it is not possible to conclude from this study that this issue could be mitigated by the optimization of their haemoglobin levels, without also addressing other potential aetiological causes. Further research into such causes, their relative mutual importance, and the development of targeted helpful interventions, should be a priority area for research going forwards.


Cancer is a generic term for a large group of diseases characterized by the growth of abnormal cells, which is the second leading cause of death globally. To treat cancer, currently, a number of anticancer drugs belonging to various classes chemically are available. The discovery of artemisinin and its derivatives such as artesunate, arteether, and artemether became a milestone in the cure for malaria. Here, we report the anti-cancer property of anhydrodihydroartemisinin (ADHA) - a semisynthetic derivative of artemisinin against prostate cancer cell line PC-3. ADHA was found to be inhibiting growth of PC-3 cells. ADHA was also found to be inhibiting migration of PC-3 cells. At molecular level, ADHA was found to be inhibiting the expression of c-Jun, p-c-Jun, p-Akt and NF-κB and activated caspase 3 and 7. The results show that ADHA like few other artemisinin derivatives hold potential to be used as an anti-cancer agent against prostate cancer cells.  Artemisia annua L. (Qinghao). ART, also known as Qinghaosu, was discovered by Tu Youyou in the early 1970s and the structure of ART was determined in 1979  is basically a natural endoperoxide containing sesquiterpene isolated from one kind of Chinese medicinal plant al., 2014; Zhu et al., 2014; Weatheral., 2014). ART isal., 2014). Recental., 2014; Button al., 2014; Lai et al., 2014). The moal., 2014).Since the discovery of ART, efforts have been made on the chemical modifications of it to improve the pharmaceutical value . Thus, PC-3 cell line was purchased from the National Center for Cell Science, Pune, India and was cultured in RPMI-1640 (Invitrogen) supplemented with 10,000 units/mL penicillin and 10 mg/mL streptomycin (HIMEDIA) and 10 % heat-inactivated FCS (Invitrogen). The antibodies against c-Jun and P-c-Jun were purchased from Abcam. The anti-caspase 3, anti-caspase 7, anti-NF-κB and anti-p-Akt were purchased from Santa Cruz. Bio-Rad Clarity™ Western ECL substrate was purchased from Bio-Rad Laboratories. ADHA was obtained from National Institute of Pharmaceutical Education and Research, Mohali from Prof. KPR Kartha’s Laboratory.in vitro cell viability was evaluated by crystal violet (CV) assay. Briefly, cells were seeded in 96-well plate; 5000 cells per well followed by exposure to ADHA for 72 h in a dose-dependent manner . This incubation period was followed by removal of media and staining of cells for 30 min with 0.4 % crystal violet. The wells were washed to remove excess dye and then the plate was allowed to dry overnight. Next day, dye was dissolved in methanol and absorbance was measured at 540 nm.The Colonogenic assay was done with PC-3 cells. For the assay, 500 cells/well were plated in the six-well plate. Once the cells were attached, cells were exposed to ADHA in a dose-dependent manner. After that, the cells were fixed with methanol and stained with 0.4 % crystal violet and colonies were counted using imageJ software.5 PC-3 cells were seeded in each well of a six-well plate and the cells were allowed to form a monolayer. The monolayer so formed was given a scratch to create a gap. The movement of cell front was photographed at 0 and 12 h. Analysis of the wound area was done by imageJ software.Approximately 3 X 10PC-3 cells treated with ADHA were harvested after a 72 h incubation period and lysed in 2X SDS lysis buffer containing 0.5 M Tris-HCl, pH 6.8, glycerol, 10 % (w/v) SDS, protease inhibitor cocktail. The lysates were sonicated and centrifuged at 16000 g for 20 min following which supernatant was collected and subjected to protein estimation by BCA method. 30-40 µg of proteins were separated by SDS gel and transferred onto PVDF membranes. Membranes were blocked with 5 % skim milk followed by incubation with primary antibodies kept at 4 ºC for overnight and with secondary antibody for 1 h. Proteins were detected using chemiluminescence.The results are expressed as the mean ± standard deviation (SD). A paired t-test was used to determine the significant difference between groups. A p-value < 0.05 was considered as significant.PC-3 cells treated with ADHA were subjected to crystal violet assay. As shown in Figure 2ASince PC-3 cells are metastatic in nature, we checked the anti-migratory property of ADHA. Our result showed that ADHA significantly inhibited the migration of PC-3 cells at both 3 and 10 µM as shown in Figure 3AκB and checked the expression of markers associated with intrinsic mode of apoptosis. We found that ADHA has a suppressive effect on the expression of the c-Jun, p-c-Jun and NF-κB particularly at 10 µM as shown in Figure 4AWe studied the effect of ADHA on the expression of c-Jun, p-c-Jun and NF-Artemisinin derivatives are being studied worldwide for their efficacy against a number of diseases, primarily for malaria. We here, report the anti-cancer effect of an artemisinin derivative; ADHA. Although the anti-leishmanial and anti-malarial activity have been reported for the ADHA , its anIn view of identifying the molecular markers, we checked the expression of c-Jun and phospho-c-Jun, the latter is a positive regulator of cell cycle. It can promote cell growth and can also transduce a mitogenic response. It also plays a role in cell proliferation, migration and invasion of cancer (Zhang et al., 2007; Mialon Next, we checked a time-dependent expression of phospho-Akt which is a proven survival factor and can exert anti-apoptotic effects by preventing the release of cytochrome c from mitochondria. It phosphorylates and inactivates the proapoptotic factor BAD and procaspase 9. And, also it phosphorylates and inactivates the transcription factor FOXO which regulates the expression of genes important for apoptosis (Altomare and Testa, 2005; Kumar eAgain, NF-κB plays a crucial role in cancer initiation and progression. It regulates a variety of cellular processes including cell proliferation. It plays a role in cancer progression by regulating the epithelial to mesenchymal transition (Hoesel and Schmid, 2013; Xia et Finally, we can conclude that ADHA has cytotoxic effects on PC-3 cells and causes cell death via apoptosis of PC-3 cells via caspase-dependent intrinsic signaling cascade.Faiz Ahmad and Amit Sarder contributed equally to this manuscript.


Osteosarcoma is the most common primary malignant tumor, especially in children and adolescents. Circular RNAs (circRNAs) are found to play roles in the progression of osteosarcoma. However, the exact functions of circRNAs in osteosarcoma development still need to be clarified. We obtained differentially expressed circRNAs and miRNAs from a GSE99671 data set (GEO database). The gene co-expression network of ceRNAs and osteosarcoma-related genes was analyzed using the STRING database. qRT-PCR was used to detect the expression of circ-03955 and miR-3662. Transwell assays and flow cytometry were performed to detect phenotypic changes in cell function. A xenograft tumor model was established using BALB/c nude mice. Dual luciferase activity and RNA immunoprecipitation assays were performed to assess the relationship between circ-03955, miR-3662, and metadherin (MTDH). Immunohistochemistry, immunofluorescence, and Western blotting were used to assess protein expression levels. Circ-03955 was significantly upregulated, and miR-3662 was downregulated in osteosarcoma. Circ-03955 silencing inhibited the growth and metastasis of osteosarcoma. Mechanism analysis revealed that circ-03955 could bind to miR-3662, and the latter could target MTDH, leading to its suppressed expression and facilitating epithelial-mesenchymal transition (EMT). All these findings demonstrate that the presence of circ-03955 promotes EMT in osteosarcoma by acting as miR-3662 sponge-mediated MTDH expression. Osteosarcoma is the most common malignant bone neoplasm, especially in children and adolescents. It has a high rate of mortality and disability , 2 as weCircular RNA (circRNA) is a relatively newly identified form of RNA. The 3’ and 5’ ends of circRNA are linked together, forming a closed ring structure, and circRNA is abundant in the eukaryotic transcriptome. In 2012, Salzman , 7, tumoCurrent studies show that miRNAs can be involved in the regulation of gene expression and can complement the transcripts of target genes, thus playing an important role in the occurrence and development of tumors , 17. TheIn this study, we first screened circRNA (circ-03955) from the GEO database (GSE99671 data set) and find that circ-03955 was highly expressed in osteosarcoma. We also find that circ-03955 knockdown could inhibit the migration, invasion, and epithelial-mesenchymal transition (EMT) of osteosarcoma. In addition, circ-03955 acts as a ceRNA for miR-3662 to regulate the progression of metadherin (MTDH)-mediated osteosarcoma.Forty samples of osteosarcoma tissue and 40 cases of adjacent non-osteosarcoma tissues were obtained from the Department of the third Xiangya Hospital of Central South University. All tissue specimens were removed during scheduled surgery. After rinsing the tissue specimens with saline, the specimens were cut into small pieces and frozen in liquid nitrogen. The specimens were then stored at −80°C for subsequent RNA extraction experiments. The use of normal bone tissue specimens and osteosarcoma bone tissue specimens in all patients was evaluated and approved by the Ethical Committee of the third Xiangya Hospital of Central South University. All participants provided written informed consent after receiving information about the design of the study.A human osteoblastic cell line, OB3, and the human osteosarcoma cell lines Saos-2, MG-63, and U2OS were purchased from the American Type Culture Collection . Saos-2 and U2OS cells were cultured in McCoy’s 5a medium modified , but 15% fetal bovine serum was added to Saos-2 growth medium, and 10% fetal bovine serum was added to MG-63 growth medium. MG-63 cells were cultured in Eagle’s minimum essential medium  containing 10% heat-inactivated fetal bovine serum. The circ-03955 interference vectors sh-circ-03955-1 and sh-circ-03955-2 and the circ-03955 overexpression vector pCDNA3.1-circ-03955 (OE-circr-03955) were obtained from GenePharma . A non-targeting shRNA (shNC) or the empty pcDNA3.1vector (OE-NC) were used as negative controls. The miR-3662 mimics and miR-3662 inhibitor were purchased from GeneCopoeia . The MTDH mRNA interference vector sh-MTDH was obtained from GenePharma . All transfections were performed using Lipofectamine 6000 reagent  according to the manufacturer’s protocols.https://www.ncbi.nlm.nih.gov/geo) was analyzed for differential gene expression. Gene expression was calculated using | log2FC | > 1 and FDR < 0.05 as the criteria for significant difference using the R statistics package limma. The miRanda package was used to predict the miRNAs that can bind MTDH and circRNA, and then the possible ceRNA network of differentially upregulated circRNA, differentially downregulated miRNAs, and MTDH was constructed. The first 20 genes reported to be associated with osteosarcoma were selected from the DisGeNET database for co-expression analysis with the ceRNA network maps. Cytoscape 3.6.1 was used to build the network graph.The GSE99671 data set, which included 18 pairs of osteosarcoma tissue specimens and paired normal bone tissue specimens, from the GEO database , primer circ-03955 , primer miR-3662 , U6 , and the internal reference primer GAPDH . All primers were designed and synthesized by Sangon Biotech .The linear validation of circRNA was performed in the osteosarcoma cell line MG-63. MG-63 cells were extracted using Trizol reagent , and circRNA was linearized by RNase R. Then, the expression of circular RNA was detected using qRT-PCR assay. The specific steps were carried out as previously described . SYBR Gr5/well. The culture plate was placed in an incubator at 37°C containing 5% CO2 and saturated humidity. The suspension of cells was centrifuged at 2000 rpm for 15 minutes. The supernatant was dissolved and crystallized by adding 150 μL DMSO. The cells were transfected for 24 hours by enzyme-linked immunosorbent assay . The OD value at 570 nm was detected after 24, 48, and 72 hours of transfection.Cells were digested with 0.25% trypsin and suspended in basic culture medium containing 10% fetal bovine serum to form a single-cell suspension. The single-cell suspension was inoculated into 96-well plates at a concentration of 1 × 106/mL) in basic culture medium containing 10% fetal bovine serum. After centrifugation with 1000 rpm for five minutes, the culture medium was discarded, the cells washed with precooled PBS  and centrifuged at 1000 rpm for 5 minutes. The cells were resuspended in 100 μL of binding buffer (1 × 105/mL), lightly blended with 5 μL Annexin V-FITC at room temperature for 10 minutes and then incubated with 5 μL PI at room temperature for 5 minutes. PBS was then added to 500 μL, and the apoptotic rate was detected immediately after blending.The cells were digested with 0.25% trypsin, and then the digested cells were combined into a single-cell suspension  was added to the bottom of the upper chamber and incubated in a 37°C incubator for 4 hours after dilution by 100 μL. The gelatinized material was then removed and reserved.Serum was removed to starve the cells for 24 hours, and 0.25% trypsinase was added to digest the cells. After termination of digestion, the cells were washed twice with precooled PBS and then suspended in serum-free medium containing BAS, and the cell concentration was adjusted to 5 × 105 cells. After the osteosarcoma MG-63 cells were injected into the mice, the tumor volume was measured using calipers every five days. Six weeks later, the mice were euthanized, and the tumors were weighed. The study was approved by the medical ethics committee of the third Xiangya Hospital, central South University.Ten 4- to 5-week old SPF male BALB/c nude mice were purchased from Auragene . Nude mice were randomly divided into an sh-NC group and an sh-circ-03955 group with five mice in each group. Osteosarcoma MG-63 cells transfected with sh-circ-03955 and sh-NC plasmids were administered by subaxillary injection to form tumors at a cell concentration of 2 × 10Logarithmic-stage growth cells of different treatment groups were washed twice with PBS and then used to prepare cell smears. The cells were fixed at room temperature with 4% paraformaldehyde for 30 minutes, washed twice with PBS, and then treated at room temperature with 0.1% Triton-X 100 for 3 minutes to make them permeable. After washing the permeate, 5% skimmed milk powder was added and incubated at room temperature for 2 hours. Anti-E-cadherin antibody  and anti-N-cadherin antibody  was added and the reaction incubated overnight at 4°C. After the first antibody was washed out, fluorescently labeled goat antimouse IgG antibody  was added and incubated at room temperature for 1 hour, then washed with PBST three times. After adding DAPI staining and washing out the staining solution the cells were observed under the fluorescence microscope .Tissues or cells were removed, and then total protein was extracted using RIPA buffer. BCA protein concentration kits  were used to determine the protein concentration. SDS sample buffer was added, and the reaction mixture was placed in a 95°C water bath for 5 minutes, then centrifuged after cooling. Electrophoresis using 10% SDS-PAGE gels was performed with 20 μg protein per well. After the completion of electrophoresis, the protein obtained was transferred to PVDF transfer membranes using a wet rotation method. The protein was sealed at room temperature for one hour with 5% BSA . Then antibodies to MTDH , E-cadherin , N-cadherin , Vimentin , GAPDH , and β-actin  were added to the membrane. After incubation overnight at 4°C, the membranes were washed three times with TBST buffer. Membranes were incubated at room temperature for 30 minutes with goat antimouse IgG antibody labeled with horseradish peroxidase and then washed with TBST buffer three times. Color was developed using immunoblotting chemiluminescence reagent ECL  and then detected using a gel imaging system.2O2-methanol solution for 15 minutes at room temperature to block endogenous peroxidase and then washed with PBS three times. E-cadherin  and N-cadherin  were added and incubated in a 37°C water bath for one hour, followed by post-blocking incubation at room temperature for 30 minutes and then washing with PBS three times. The second antibody labeled with horseradish peroxidase was incubated at room temperature for 30 minutes and washed with PBS five times. After adding DAB for two minutes, the dyeing was terminated, re-dyed with hematoxylin for three minutes, and then sealed with transparent xylene and dripped with neutral gum. Under the light microscope, positive brown staining was observed at a magnification of 200×.Immunohistochemistry (IHC) was performed to evaluate changes in the expression levels of E-cadherin and N-cadherin. The xenograft tissue sections of the sh-NC and sh-circ-03955 groups were taken out and completely dewaxed and hydrated. The antigens were repaired by microwave. Then, the sections were washed with PBS three times. The sections were immersed in 3% H® Reporter Assay System . MTDH binding to miR-3662 was detected in a similar way.MG-63 cells were transfected with Wt-circ-03955 plasmid and NC mimics or miR-3662 mimics, according to the instructions accompanying the Lipofectamine 6000 transfection reagent. Mut-circ-03955 plasmid and mimics of NC or miR-3662 were co-transfected, and lysate was added 48 hours later. The supernatant was centrifuged for 10 minutes at 4°C at 12,000 rpm, and the luciferase activity was detected using a Dual-Luciferase7) were collected and added to the same volume of RIP lysate to lyse cells. The supernatant was centrifuged at 12,000 rpm for 10 minutes. In accordance with the instructions of the RIP™ RNA binding protein immunoprecipitation reaction kit, 900 μL of RIP immunoprecipitation buffer containing RNase inhibitor, protease inhibitor, and DNase and 100 μL of cell lysate were added into EP tubes containing magnetic beads. IgG antibody or Ago2 antibody were added and incubated overnight at 4°C and centrifuged at 12,000 rpm. After centrifugation for 10 minutes, the supernatant was discarded and washed six times with 500 μL RIP wash buffer. The RNA was purified immediately, and then dissolved and purified in 15 μL of DEPC water, then stored at −80°C. The positive control (input) group had no antibody, and the experimental group had Ago2 antibody. The expression of circ-03955 and microRNA-3662 in the input group was detected using qRT-PCR assay.MG-63 cells . Chi-square tests were used to analyze the correlations between the expression of miR-3662 and circ-03955 in osteosarcoma cells. The P value was evaluated to determine statistical significance with a P value of less than 0.05 taken to indicate that the difference was statistically significant.SPSS19.0 statistical software and GraphPad Prism 6.0 software were used to analyze all the data. Student’s To screen differentially expressed miRNA and circRNA in osteosarcoma, we performed bioinformatic analysis on data from the GSE99671 data set, comprising 18 pairs of osteosarcoma tissue specimens and paired normal bone tissue specimens. We identified nine downregulated miRNAs Table 1 To verify the differential expression of circ-03955 and miR-3662 in osteosarcoma tissues and cells, we first linearized them using RNase R in MG-63 cells, and then detected the expression of circ-03955. The results show that circ-03955 is resistant to RNase R digestion, and linear circ-03955 is easy to degrade. They further confirm that circ-03955 is circular (P < 0.001) (P < 0.001) (P < 0.001)   (P < 0.001)  (P < 0.001) (P < 0.001) . To verify the effect of circ-03955 on the expression of miR-3662 and MTDH, OE-circ-03955 or sh-circ-03955 were transfected into MG-63 cells. The qRT-PCR and Western blot results showed that circ-03955 overexpression can inhibit the expression of miR-3662 and promote the expression of MTDH. The knockdown of circ-03955 was shown to promote the expression of miR-3662 and inhibit the expression of MTDH . The RIP test was performed with Ago2 as the antibody to research the relationship between circ-03955 and miR-3662. We found that circ-03955 and miR-3662 were preferentially enriched in the Ago2 pellet relative to control IgG immunoprecipitates, and endogenous circ-03955 pull-down was specifically enriched in miR-3662-transfected cells  can be found in the article/supplementary material.The animal study was approved by the medical ethics committee of the third Xiangya Hospital, central South University.YZ designed the work and offered funds. ZW and MD designed the work, performed the experiment, and submitted the manuscript. LC and WW performed the experiment and collected the data. GL, DL, and ZH analyzed data and modified the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by the New Xiangya Talent Project of the Third Xiangya Hospital of Central South University (Grant NO. JY201516).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Urine cotinine-measured SHS exposure was examined to determine the exposure level. A cross-sectional study including 17 children in a low-exposure group, 17 in a medium-exposure group, and 17 in a high-exposure group was first conducted. Then, a before–after study in the cohort of children was carried out before and two months after smoking-cessation intervention for family smokers. A total of 43 metabolites were discovered to be related to SHS exposure in children in the cross-sectional analysis  corrected p < 0.05, variable importance in the projection (VIP) > 1.0). Only three metabolites were confirmed to be positively associated with children’s exposure to SHS (FDR corrected p < 0.05) in a follow-up longitudinal analysis, including kynurenine, tyrosyl-tryptophan, and 1-(3-pyridinyl)-1,4-butanediol, the latter of which belongs to carbonyl compounds, peptides, and pyridines. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that 1-(3-pyridinyl)-1,4-butanediol and kynurenine were significantly enriched in xenobiotic metabolism by cytochrome P450 (p = 0.040) and tryptophan metabolism (p = 0.030), respectively. These findings provide new insights into the pathophysiological mechanism of SHS and indicate the influence of individual differences in SHS-induced changes in urinary metabolites in children.Children’s exposure to secondhand smoke (SHS) is a severe public health problem. There is still a lack of evidence regarding panoramic changes in children’s urinary metabolites induced by their involuntary exposure to SHS, and few studies have considered individual differences. This study aims to clarify the SHS-induced changes in urinary metabolites in preschool children by using cross-sectional and longitudinal metabolomics analyses. Urinary metabolites were quantified by using untargeted ultra high-performance liquid chromatography-mass spectrometry (UPLC Children’s exposure to secondhand smoke (SHS) is a major public health problem, with a prevalence of 40% worldwide and 55.9% in Southeast Asia ,2. In ChTobacco smoke contains more than 4000 kinds of constituents, including nicotine, tar, carbonic monoxide, polycyclic aromatic hydrocarbons, and heavy metals . NicotinMetabolomics has emerged as a powerful tool for understanding metabolic changes in response to pathophysiological conditions or environmental exposures, providing an opportunity to identify biomarkers of exposure to tobacco smoke and markers that reflect host-related metabolic adaptations . SeveralSubject recruitment was carried out in the Hetou and Luoyang village communities, Taizhou city, Zhejiang province, China, from 15 March 2018 to 31 March 2018. The cluster sampling method was used to select the Hetou and Luoyang village communities. Smokers were identified using the health records of community health service stations. Study coordinators went to each smoking family to describe the study. Smoking families were enrolled if they met the following criteria: (1) Smokers reported having children aged 2–5 years and smoking at home and in front of children; (2) caregivers of children were willing to provide the children’s urine samples; and (3) both the smokers and the caregivers of children provided written informed consent. At the baseline, data collection was performed via an in-person questionnaire survey administered to smokers and caregivers. Data included the smoking site/venues, the smoking frequency in the past week, and the average daily cigarette amount in the past week for smokers, and the health condition of and recent respiratory symptoms among children.The urine samples from each child were collected in sterile fecal collection containers at home or at a community health service station and then placed in a portable refrigeration apparatus (−20 °C) immediately after sampling. Upon receipt, the urine samples were immediately frozen and stored at −80 °C until analysis.This study was approved by the ethics committee of Duke Kunshan University (IRB No: 2016ABDU003). Written informed consent was obtained from each smoker and caregiver of the child.From 15 May 2018 to 15 July 2018, we conducted smoking-cessation interventions for smokers, including health education counseling delivered by community health workers in five different sessions (two in-person and three via-telephone sessions). The counseling covered hazards about smoking and SHS exposure, tips on quitting smoking and overcoming withdrawal syndrome, how to prevent relapse, and how to ensure a smoke-free home. Follow-up interviews and assessments were conducted at 2 months after the intervention for smokers and caregivers of children. Urine samples were also collected at the follow-up.Urine cotinine was analyzed using a liquid chromatography tandem mass spectrometry (LC-MS) method. Urine cotinine levels were reported in nanograms per milliliter and standardized per milligram of creatinine. Cotinine concentrations below the limit of detection  were considered unobservable and inaccurate ,29, and SHS exposure was determined by smokers’ self-reports and by measuring the children’s urinary cotinine concentrations. Positive SHS exposure was defined when both the self-reported SHS exposure in children by smokers and urine cotinine-measured SHS exposure were positive.Children were excluded if they met the following criteria: (1) Lost to follow-up; (2) those with missing laboratory measurements of the urinary cotinine concentration; and (3) those with a cotinine concentration at the boundary value according to the cutoff value of group stratification.v/v) [v/v) formic acid) and solvent B (acetonitrile (0.1% (v/v) formic acid) at a flow rate of 0.40 mL/min. The separation was achieved with the following elution gradient: 1% B over 0–1 min; 1% B-20% B over 1–5.5 min; 20% B-30% B over 5.5–6 min; 30% B-35% B over 6–8.5 min; 35% B-70% B over 8.5–10.5 min; 70% B-100% B over 10.5–11 min; and the composition was held at 100% B for 2 min, and then 13–13.1 min, 100% B to 1% B, and 13.1–15 min holding at 1% B. The MS signal acquisition was performed in positive and negative ion scanning modes. To obtain information regarding the system repeatability, QC samples were injected at regular intervals  throughout the analytical run [Urine samples (100 μL) were added to 300 μL methanol-water  . Each saical run .(c)-MS/MS raw data were processed using Progenesis QI software , and a data matrix containing the retention time, mass-to-charge ratio, and peak intensity was obtained. The data matrix was preprocessed as follows: By (1) retaining variables with >50% of nonzero values in all samples [http://www.hmdb.ca/) and Metlin database (https://metlin.scripps.edu/). Concretely, the mass tolerance between the measured m/z values and the exact mass of the components of interest was ±10 ppm. For metabolites having MS/MS confirmation, only the ones with MS/MS fragment scores above 30 were considered as having been confidently identified; otherwise, metabolites only had tentative assignments. The positive and negative data were combined and imported into the SIMCA-P + 14.0 software package . Data analysis was performed on the Majorbio I-Sanger Cloud platform (www.i-sanger.com).UPLC samples ; (2) fil samples ; (3) nor2Y and Q2 were used to evaluate the goodness-of-fit and predictive ability of each model. Through an analysis of OPLS-DA loadings, taking the BL group as a reference, an independent-sample hypothesis test was used for the BH and BL groups, as well as for the BM and BL groups, in the cross-sectional study. The differential metabolites were identified with a variable importance in the projection (VIP) of greater than 1.0 and false discovery rate (FDR) corrected p values of less than 0.05 (Student’s t-test using the stats package in R). A cluster heatmap of the metabolites identified in this process was constructed using the Pheatmap package in R. The paired-sample hypothesis test (paired t-test using the stats package in R) was used to verify the differential urinary metabolites through a comparison of before intervention (pre-intervention) and after intervention (post-intervention) paired-samples from each child in the ID and INC group in the longitudinal study. Significant differences were considered when results were below an FDR threshold of 0.05. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database and HMDB database were also used to uncover the predicted molecular pathways and biological functions of the metabolites. KEGG enrichment analysis of common target metabolites was performed using Fisher’s exact test to obtain the significantly enriched pathways [www.i-sanger.com).Orthogonal partial least-squares discriminant analysis (OPLS-DA) was performed using the ropls package in R to visually discriminate between BL and BH groups, as well as between BL and BM groups at the baseline. This approach aims to maximize the covariance between the outcome and matrix of metabolites by projecting both to linear subspaces of the original variables ,36. Valipathways . All of t-test. Categorical variables were described with numbers and percentages, and were analyzed by Fisher’s exact test in terms of the three baseline SHS-exposure level groups and McNemar’s test for the pre-intervention and post-intervention groups. All tests for significance were two-sided, and p-values < 0.05 were considered significant.Analyses of demographic data were carried out using SPSS software. . Continuous variables were described with the mean and standard deviation (SD), and were compared with regards to the three baseline SHS-exposure level groups by using analysis of variance (ANOVA) and the pre-intervention and post-intervention groups by using a paired Student’s 2 Y and Q2 indicated a good fitness, and the negative Q2 from the 200-time permutation tests suggested no overfitting in OPLS-DA models  corrected p) and multivariate OPLS-DA analysis (predicted by the variable importance in the projection (VIP)) . A total of 43 metabolites were consistently implicated in these two comparisons  and low-exposure (BL) groups, as well as between medium-exposure (BM) and BL groups . Seven-fA models . At the n (VIP)) . Taking n (VIP))  and 100 n (VIP))  were obsThe color of each section represents the abundance value of metabolite calculated by the relative quantitation normalization method. Each row corresponds to data for a specific metabolite, and each column represents the BL, BM, or BH group. Different colors represent the different intensity levels of metabolites.As shown in t-test, p = 0.004), and the smoking amount decreased from 21.38 (SD 11.17) to 12.56 (SD 6.63) . As expected, there was no significant difference in the smoking frequency and smoking amount before and after the intervention in the INC group (p > 0.050). For children’s overall health status and respiratory symptoms reported by their caregivers, there was no significant difference before and after the intervention in both the ID and INC groups.The follow-up data are presented in t-test was performed to verify the differential urinary metabolites through a comparison of pre-intervention and post-intervention paired-samples from each child in the ID group and INC group, respectively. The metabolites showing significant differences in the ID group (FDR corrected p < 0.05) while no change in the INC group (FDR corrected p > 0.05) after 2 months of intervention were finally identified as the exact urinary metabolic biomarkers of SHS exposure. Details of the paired comparison for the 43 metabolites are shown in p = 0.011 in the ID group and FDR corrected p = 1.000 in the INC group), 0.69-fold for the 1-(3-pyridinyl)-1,4-butanediol level (FDR corrected p = 0.009 in the ID group and FDR corrected p = 1.000 in the INC group), and 0.78-fold for the kynurenine level (FDR corrected p = 0.036 in the ID group and FDR corrected p = 1.000 in the INC group). Additionally, For the 43 differential metabolites implicated in the cross-sectional analysis, a paired p = 0.040), and kynurenine was significantly enriched in tryptophan metabolism (p = 0.030). As shown in Red represents the metabolites reported in the present study, which showed a positive relation with SHS exposure. The dotted arrows indicate indirect reactions. Abbreviations: IDO, indoleamine 2,3-dioxygenase; MAO, monoamine oxidase; TDO, tryptophan 2,3-dioxygenase; TPH, tryptophan hydroxylase; KMO, kynurenine 3-monooxygenase; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NNAL, 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanol; and CYP, Cytochrome P450.To the best of our knowledge, this is the first study to combine cross-sectional and longitudinal metabolomics to clarify the SHS-induced changes in urinary metabolites in children. Three metabolic biomarkers, including tyrosyl-tryptophan, 1-(3-pyridinyl)-1,4-butanediol, and kynurenine, were identified as being positively associated with cotinine exposure in children. Among these three urinary metabolites, it is the first time that tyrosyl-tryptophan and 1-(3-pyridinyl)-1,4-butanediol have been reported to be related to SHS exposure.The results from our study suggested a positive relation between kynurenine and tyrosyl-tryptophan and SHS exposure. The kynurenine pathway is responsible for tryptophan metabolism, and 95% of tryptophan is metabolized via the kynurenine pathway . TherefoWe also found a positive relation between tyrosyl-tryptophan and the cotinine concentration. Tryptophan has been extensively studied, but evidence of tyrosyl-tryptophan is sparse. Tryptophan is a nutritionally essential amino acid for both humans and animals. In addition to acting as a building block for protein synthesis, tryptophan and its metabolites are crucial for maintaining neurological function, immunity, and homeostasis in the body . PaternaN-nitrosomethylamino)-1-(3-pyridyl)-1-butanol (NNAL) pathway and serves as a downstream metabolite of NNAL. NNAL is the most potent tobacco-specific carcinogen, and it has been frequently used as a biomarker to assess human smoke exposure [Our study is the first to reveal a positive correlation between urine 1-(3-pyridinyl)-1,4-butanediol and the cotinine concentration. As shown in exposure . Both urexposure . Our datA previous study on metabolites relevant to children’s exposure to SHS mainly focused on targeted metabolomics. A randomized clinical trial in 195 female smokers with children aged ≤ 10 years residing in their homes aimed to promote smoke-free homes through biomarker feedback documenting a child’s exposure to tobacco toxins, documenting the levels of nicotine, cotinine, and NNAL . A previSeveral limitations of this study should be acknowledged. First, although we combined the cross-sectional baseline comparison with the longitudinal pre-intervention and post-intervention comparison to remove the influence of individual differences and improve the reliability of the results, this study included a preliminary analysis with a small sample size, and studies of larger populations with different groups of children across Asia or with different ethnicities would give a better picture of the connection between urinary metabolites and SHS exposure. Second, the urine samples were only collected once, and we were unable to assess the temporal trends of metabolites over time, making it impossible to derive the long-term effects of metabolites on children with SHS exposure. Subsequent studies should measure urine samples multiple times to obtain long-term influence trends of metabolites. Third, although our study suggests tyrosyl-tryptophan, 1-(3-pyridinyl)-1,4-butanediol, and kynurenine as urinary metabolic biomarkers for SHS exposure, which may be involved in the pathogenesis of disease of the respiratory system, further work, including long-term observations of children and animal experiments, will be needed to determine whether these urinary metabolic biomarkers are predictors of long-term illness. Fourthly, the study lacks urine samples from control children, who were not exposed to second-hand smoke, because this study was based on the smoking-cessation intervention program and because it is difficult to obtain preschool children’s samples and information in nonsmoking families. However, we set low, medium, and high urine cotinine levels and treated the low-concentration group as the control group to perform multiple comparison analyses, in order to address this limitation. Fifthly, we did not test the markers of kidney function in urine, which may have some influence on the level of cotinine and metabolites in urine. Further research with elaborate markers of kidney function in urine is needed to confirm the SHS-associated metabolites identified in our study. Finally, although, in metabolomics work, it is common to use a criterion of a 50% rule to retain the metabolites with missing intensity values in no more than 50% of the samples for statistical analysis , the impIn summary, this study is the first to have combined cross-sectional and longitudinal metabolomics analysis to examine urinary biomarkers in response to children’s SHS exposures. In addition to the well-established nicotine metabolite kynurenine, we newly identified tyrosyl-tryptophan and 1-(3-pyridinyl)-1,4-butanediol as potential metabolic markers and related functional pathways affected by SHS in children, contributing new insights into the pathophysiological mechanism of SHS. Importantly, our findings indicated that individual differences should be taken into account when exploring urinary metabolites related to children’s exposure to SHS.


Ipomoea obscura (L.) Ker Gawl. aqueous leaf extract. The UV-visible spectral analysis of the ZnO-NPs showed an absorption peak at 304 nm with a bandgap energy of 3.54 eV, which are characteristics of zinc nanoparticles. Moreover, the particles were of nano-size (~24.26 nm) with 88.11% purity and were agglomerated as observed through Scanning Electron Microscopy (SEM). The phyto-fabricated ZnO-NPs offered radical scavenging activity (RSA) in a dose-dependent manner with an IC50 of 0.45 mg mL−1. In addition, the genotoxicity studies of ZnO-NPs carried out on onion root tips revealed that the particles were able to significantly inhibit the cell division at the mitotic stage with a mitotic index of 39.49%. Further, the cytotoxic studies on HT-29 cells showed that the phyto-fabricated ZnO-NPs could arrest the cell division as early as in the G0/G1 phase (with 92.14%) with 73.14% cells showing early apoptotic symptoms after 24 h of incubation. The results of the study affirm the ability of phyto-fabricated ZnO-NPs from aqueous leaf extract of I. obscura is beneficial in the cytotoxic application. The study was undertaken to investigate the antioxidant, genotoxic, and cytotoxic potentialities of phyto-fabricated zinc oxide nanoparticles (ZnO-NPs) from  Nanotechnology is a multidimensional discipline that has the potential to transform all the fields of science. Nanoparticles have gained importance due to their surface area to volume ratio and the behaviors that result from that ,2. In liIt has been well documented in the literature that the chemical synthesis of nanoparticles yields toxic byproducts that are not eco-friendly and cost-effective, and researchers are concentrating on alternative methods ,5,6. BioIpomea spp. have been in continuous use in nutrition, medicine, rituals, and agriculture [Ipomea spp. which have been evaluated for antioxidant and cytotoxic effects [Ipomea obscura has been made. Hence, a study on antioxidant, genotoxic, and cytotoxic potentialities of phyto-fabricated zinc oxide nanoparticles (ZnO-NPs) from I. obscura aqueous leaf extract was evaluated.iculture . These piculture ,20. From effects ; howeverIpomea obscura (L.) Ker Gawl. were collected from Manasagangotri, Mysuru, Karnataka, India and authenticated at the Dept. of Studies in Botany, University of Mysore, Mysuru. Phyto-fabrication of ZnO-NPs from I. obscura leaves was carried out according to our previous studies with modifications [Healthy leaves of ications . In brieI. obscura was investigated by FT-IR with a resolution of 4 cm−1 between 4000 to 400 cm−1 spectral range on a PerkinElmer Spectrum ATR2000 (Singapore).The UV-Vis Spectrophotometer  was used to determine the optical density of the phyto-fabricated ZnO-NPs. X-Ray Diffraction (XRD) study was performed out on an X-Ray Diffractometer , and Scherrer’s formula was applied to determine the particle size. The Dynamic Light Scattering (DLS) analysis was also carried out to learn the particle size distribution of ZnO-NPs using the Nanotrack Wave particle size analyzer . The morphology of the nanoparticles was evaluated by Scanning Electron Microscopy  at 5 kV acceleration. The phyto-fabricated ZnO-NPs were placed on a carbon-coated copper in a tiny amount, allowed to air-dry, and images were taken. The elemental analysis  was carried out by Energy Dispersive Spectroscopy (EDS) . The binding properties of the ZnO-NPs and aqueous extract of −1 concentrations of ZnO-NPs were sonicated before incubating for 30 min at 37 ± 2 °C (under dark conditions) and the absorbance was measured at 517 nm in a spectrophotometer. The percentage of RSA of the nanoparticles was determined.The radical scavenging activity (RSA) of phyto-fabricated ZnO-NPs was performed by following the method of Hemanth Kumar et al. . In brie−1) and were subjected to incubation for 24 h at room temperature. The ZnO-NPs treated onion root tips were watchfully cut out and fixed in Carnoy’s Fixative II (for 24 h) and later relocated in 70% ethanol. Later, the root tips were taken and squashed, and the cells were observed for cell division and, also, to record the mitotic index. Sterile Distilled Water (SDW) and Methotrexate treated root tips were designated as the negative and positive control, respectively. A total of ten microscopic fields per root sample were observed with four roots per treatment.The genotoxicity of ZnO-NPs was determined in the root tips of healthy onion bulbs . Onion b5) in Dulbecco’s modified Eagle’s medium (DMEM) and further incubated for 24 h at 37 °C (humidified atmosphere containing 5% CO2). After incubation, the media were discarded, and each well was loaded with 150 µL of fresh DMEM media containing ZnO-NPs  with fetal bovine serum (10%) and incubated for 24 h. The incubated samples were aspirated and 100 µL of MTT solution (0.5 mg mL−1 in 1× PBS filtered through 0.2 µM filter) was added and subjected to incubation for 4 h. From the incubated samples, MTT reagent was removed and DMSO (100 µL) was added to solubilize formazan rapidly. SDW and colchicine treated cells were designated as a negative and positive control, respectively. Each sample was subjected to UV-Vis spectroscopy at 570 nm, and the percentage of cell viability was calculated.MTT assay was carried out to identify the anticancer properties of phyto-fabricated ZnO-NPs according to the method of Selvakumaran et al. . Cell li−1 of HT-29 cell suspension (5 × 105) was loaded into each well of a 6-well plate and subjected to incubation at 37 °C for 24 h (5% CO2). After incubation, the growth media were discarded by aspiration and reloaded with 1 mL of phyto-fabricated ZnO-NPs (1 mg) and colchicine (320 µg) before subjecting to incubation in humified conditions. The treated cells were detached by trypsinization and subjected to centrifugation for 10 min at 2000 rpm and repeatedly washed with PBS. Later, the cell pellet was fixed with ethanol (700 µL) and further incubated (for 1 h at −20 °C). Subsequently, the cells were washed twice with PBS (ice cold) by cold centrifugation for 10 min at 4000 rpm. The resultant cell pellet was resuspended in PBS (1 mL) containing propidium iodide (50 mg mL−1), RNase A (50 mg mL−1), and Triton X-100 (0.1%) and incubated for 30 min under dark conditions before analyzing using a flow cytometer .About 3 mL wellThe phyto-fabricated ZnO-NPs treated (1 mg) and respective control cells, as mentioned above, were subjected to AnnexinV-FITC staining by using Annexin V-FITC Apoptosis Detection Kit  following the manufacturer’s protocol. In brief, the ZnO-NP treated and untreated cells were resuspended in binding buffer (100 µL) containing PI (10 µL) and subjected to incubation for 5 min (dark conditions). The stained cell samples were further subjected to analysis by a flow cytometer .5 cells mL−1) and incubated for 24 h. The HT-29 cell suspension was discarded after incubation by aspiration and loaded with 1 mL of fresh DMEM media containing ZnO-NPs (1 mg mL−1). The treated samples were incubated at room temperature for 24 h aseptically. After incubation, the cellular morphology changes were observed using phase-contrast inverted microscopy at 20X magnification . Morphological changes in HT-29 cells upon treatment with phyto-fabricated ZnO-NPs were performed on a 6 well-plate. Each well was seeded with 1 mL of DMEM media containing HT-29 cells  using SPSS, version 17 . The significant differences between the treatment mean values were determined by the Honestly Significant Difference (HSD) obtained by Tukey’s test at −1 in ZnO-NPs  of 0.45 mg mL−1  and the United Nations Environmental Programme (UNEP) [A. cepa root tips, and the results of the study are presented in −1 concentration [e (UNEP) . The antntration . It has ntration ,29.I. obscura was evaluated by MTT assay against HT-29 cells. The assay studied is considered to be one of the most reliable methods to primarily assess the effect of compounds on cell lines [−1) with an IC50 value of 0.91 mg mL−1. The standard drug colchicine offered around 70% cytotoxicity at 100 µg mL−1. A significant rise in the percent cell inhibition was observed in a dose-dependent manner compared to control ; in brief, in the G1 phase, RNA and proteins are produced for the DNA replication, the S phase includes DNA synthesis, and in the G2 phase, new proteins are synthesized for cell division, whereas in the M stage, nuclear and cytoplasmic divisions take place . It is wThe biochemical hallmarks of cancer include cleavage of intracellular substrates, DNA fragmentation, and phosphatidylserine (PS), which is actively localized on the inner leaflet of the plasma membrane in healthy cells . The fliLaurus nobilis. These changes in the morphology in cell lines upon treatment with ZnO-NPs are correlated to the intracellular ROS generation, dysfunction of mitochondria, and leakage of plasma membrane leading to cell death [The morphological changes within the cells were evaluated to authenticate the results obtained from the cytotoxic studies. It was observed that ZnO-NPs and colchicine treated HT-29 cells showed morphological changes such as blebbing in the cell membrane, shrinkage of cells, the formation of apoptotic bodies, nuclear fragmentation, and loss of membrane stability E,F. In cll death . From thll death . The resI. obscura leaf extract was carried out for the first time in the present study. The physico-chemical characterization of the nanoparticles possessed bandgap energy of 3.54 eV, ~24.26 nm size with 88.11% purity. The obtained nanoparticles were free from moisture, as evidenced through FT-IR analysis. The antioxidant study revealed dose-dependent RSA with the particles showing IC50 of 0.45 mg mL−1. Further, the phyto-fabricated ZnO-NPs showed genotoxicity and cytotoxicity against A. cepa meristem and HT-29 cells, respectively, which were comparable results to that of standard drugs used. The results affirm that the phyto-fabricated ZnO-NPs could be a potent substitute for the synthetic drug used presently for cytotoxicity.In conclusion, the process of phyto-fabrication of ZnO-NPs from


D, and average number of molecules in the focus, N. The software package PAM is used to analyze all the acquired data. The data can be used as a reference for artifact-free two-color ccRICS that contains slowly diffusing interacting molecules. Additionally, the analysis workflow described in this paper helps researchers avoid common errors during a RICS experiment.The data provided with this paper are image series of slowly diffusing GlyRa3 molecules, linked to either eGFP or mCherry fluorescent proteins, at the membrane of HEK cells, acquired on a Zeiss LSM880 confocal laser scanning microscope. Raster spectral image cross-correlation spectroscopy (RSICS) is applied to the data, a technique that exploits intensity fluctuations in confocal image series recorded using a spectral detector to study the diffusion and concentration of molecules, and interactions between them. First, spectral filters are created from reference image series containing GlyRa3 labeled with a single fluorophore. Once experimental data containing GlyRa3 labeled with both fluorophores is acquired, single images are either autocorrelated, or the cross-correlation is calculated between two images, each one containing the data for eGFP or mCherry labeled GyRa 3. Data is then fit with a one-component model assuming a two-dimensional Gaussian point spread function to obtain the diffusion coefficient,  The .tif files (GlyR_REFeGFP.tif and GlyR_REFmCherry.tif) are the spectrally weighted versions of the raw data. The .miacor files are the calculated autocorrelation (GlyR_ACF1. miacor and GlyR_ACF2. miacor) and cross-correlation (GlyR_CCF.miacor) functions from the spectrally filtered data, that can be opened in the analysis software. A stepwise RSICS protocol is explained in 22.12 with 50-nm pixel size to have multiple pixels in the point-spread function. Pixel dwell, line and image times were 8.19 μs, 4.92 ms and 1.26 s, respectively. Depending on the diffusion constant range that needs to be quantified, the optimal imaging settings can be predicted [A ZEISS LSM880 laser scanning microscope and Plan-Apochromat 63x/1.4 Oil DIC M27 oil objective was used to image the HEK293 cells. For each measurement at least 60 frames were acquired at the cell membrane. Images contain 256 http://www.cup.uni-muenchen.de/pc/lamb/software/pam.html or hosted in Git repositories under http://www.gitlab.com/PAM-PIE/PAM and http://www.gitlab.com/PAM-PIE/PAMcompiled.All analyses of the experimental data were performed in the software package PAM . The sofA flowchart for the applied ccRICS, analysis using spectral weighting is shown in 2.2The spectra of the experimental single-color references data (REFeGFP.czi and REFmCherry.czi) are shown in 2.32) region-of-interest was excluded from further analysis if the mean intensity of the small ROI was >3 times the mean intensity of the complete photobleaching-corrected image series. Furthermore, the excluded-pixels mask was spatially filtered by a 3 × 3 median filter  C and D w2.4D, and average number of molecules in the focus, N.−3/2. The shift of the PSF between the green and red laser needed for fitting the cross-correlation function is represented by The auto- and cross-correlation functions  were fitted with a one-component model assuming a two-dimensional Gaussian point spread function to obtain the diffusion coefficient, To quantify the interaction between the green and red species, typically the